The present invention relates generally to the field of cell physiology, and more particularly, to apoptosis. Even more particularly, the present invention is related to a novel apoptosis associated protein EI24 and its corresponding gene EI24; to nucleotide sequences encoding EI24; to products and processes involved in the cloning, preparation and expression of genes and nucleotide sequences encoding EI24; to antibodies with specificity to EI24; and to diagnostic and therapeutic uses of the above.
xe2x80x9cApoptosisxe2x80x9d refers to cell suicide that proceeds by an active, physiological process (Kerr, J. F., et al., Br. J. Cancer 26:239-257 (1972); Wyllie, A. H., Nature 284:555-556 (1980)). Apoptosis plays an important role in developmental processes, including morphogenesis, maturation of the immune system, and tissue homeostasis whereby cell numbers are limited in tissues that ale continually renewed by cell division (Ellis, R. E., et al., Annu. Rev. Cell. Biol. 7:663-698 (1991); Oppenheim, R. W., et al., Neurosci. 14:453-501 (1991); Cohen, J. J., et al., Annu. Rev. Immunol. 10:267-293 (1992); Raff, M. C., Nature 356:397-400 (1992)).
In addition to its role in developmental processes, apoptosis is an important cellular safeguard against tumorigenesis (Williams, G. T., Cell 65:1097-1098 (1991); Lane, D. P. Nature 362:786-787 (1993)). Defects in the apoptotic pathway may contribute to the onset or progression of malignancies. Suppression of the apoptotic pathway(s), by a variety of genetic lesions, occurs frequently in a broad range of human tumors. In particular, loss of the p53 tumor suppressor gene function, either through deletion or mutation, occurs in more than 50% of human cancers. p53 gene function is also indicated in normal cell cycle events. Reviews of p53 function include Levine, A. J., et al., Nature 351:453-456 (1991); Hollstein M., et al., Science 253:49-53 (1991); Donehower, et al., Biochem. BioPhys. Acta 1155:181-205 (1993); Lane, D. P. Nature 362:786-787 (1993); Zambetti, et al., FASEB J. 7:855-865 (1993); and Greenblatt M. S., et al., Cancer Res., 54: 4855-4878 (1994).
p53 may exert its tumor suppressor function, at least in part, by directing cells that have sustained genomic damage to undergo apoptosis (Lowe S. W., Jacks T., Housman D. E. and Ruley H. E. (1994) Proc. Natl. Acad. Sci. USA, 91, 2026-2030). p53 is a sequence-specific DNA binding protein that functions both as a transcriptional activator and repressor (Donehower L. A. and Bradley A. (1993) Biochim. Biophys. Acta., 1155, 181-205; Prives C. and Manfredi J. (1993) Genes Dev., 7, 529-534; Fields S. and Jang S. K. (1990) Science, 249, 1046-1048; Raycroft L., Wu H. and Lozano G. (1990) Science, 249, 1049-1051). Although there is some evidence that transcription may not be required in p53-mediated apoptosis (Caelles C., Helmberg A. and Karin M. (1994) Nature, 370, 220-223), several p53-regulated genes have been identified to date (Kastan M. B., Zhan Q., El-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B. and A. J. Fornace Jr. (1992) Cell, 71, 587-597, 1992; El-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W. and Vogelstein B. (1993) Cell, 75, 817-825; Barak Y., Juven T., Haffner R. and Oren M. (1993) EMBO J., 12, 461-468; Wu X., Bayle J. H., Olson D. and Levine A. J. (1993) Genes and Dev., 7, 1126-1132; Zambetti G. P., Bargonetti J., Walker K., Prives C. and Levine A. J. (1992) Genes and Dev., 6, 1143-1152; Okamoto K. and Beach D. (1994) EMBO J, 13, 4816-4822; Buckbinder L., Talbott R., Seizinger B. R. and Kley N. (1994) Proc. Natl. Acad. Sci. USA, 91, 10640-10644, and two of these genes, bcl-2 and bax (Miyashita T. and teed J. (1995) Cell, 80, 293-299; Miyashita T., Krajewski S., Krajewska M., Wang H., Lin H., Hoffman B., Lieberman K. and Reed J. (1994) Oncogene, 9, 1799-1805; Zhan Q., Fan S., Bae I., Guillouf C., Liebermann D. A., O""Connor P. M. and A. J. Fornace Jr. (1994) Oncogene, 9, 3743-3751), have been clearly implicated in apoptosis (Oltvai Z. and Korsmeyer S. (1994) Cell, 79, 189-192).
In addition to cancer, deregulation of apoptosis may contribute to a number of other human diseases. A variety of degenerative disorders may involve aberrant apoptosis, resulting in premature or inappropriate cell death (Barr, P. J., et al., Biotechnology 12:487-493 (1994)) Productive infection by certain viruses may depend on suppression of host cell death by anti-apoptotic viral gene products (Rao, L., et al., Proc. Natl. Acad. Sci. USA 89:7742-7746 (1992); Ray, C. A., et al., Cell 69:597-604 (1992); White, E., et al., Mol. Cell. Biol. 12:2570-2580 (1992); Vaux, D. L., et al., Cell 76:777-779 (1994), and inhibition of apoptosis can alter the course (i.e. lytic vs. latent) of viral infection; Levine, B., et al., Nature 361:739-742 (1993)). Widespread apoptosis of T lymphocytes triggered by HIV infection may, at least in part, be responsible for the immune system failure associated with AIDS (Gougeon M., et al., Science 260:1269-1270 (1993)).
The ability of p53 to suppress tumorigenesis appears linked to its activity as a transcriptional activator, since tumor-derived mutant p53 molecules almost invariably have lost transactivation potential (Kern, S. E., et al., Science 256:827-830 (1992)). Thus, the function of the p53 tumor suppressor appears to depend, at least in part, on the ability to activate the expression of one or more target genes. Genes activated by p53 may in turn mediate one or more aspects of p53""s tumor suppressor function, which including cell cycle arrest and apoptosis, depending on the cellular context. Consistent with the notion, certain p53-activated Menses identified to date have been implicated in cell cycle control (gadd45, cyclin G, p21/WAF) and at least one p53-activated gene (bax) is linked to the regulation of apoptosis.
Tumor cells frequently have lost wild-type p53 function. Consequently, activation of p53 target genes and associated tumor suppressor functions, such as cell cycle arrest and apoptosis, is defective in cancer cells. Therefore, from the perspective of pharmaceutical development, identification of genes which are regulated (e.g., induced or repressed) by p53 may permit development of agents that activate, restore or suppress p53-dependent tumor suppression functions such as apoptosis or cell cycle regulation, depending on the clinical setting.
In a broad aspect, the present invention is directed to a gene, termed EI24, whose RNA was induced in NIH3T3 cells following treatment with etoposide. Experiments performed with cells derived from p53-deficient mice demonstrate that induction of the EI24 mRNA is dependent on expression of functional p53 in vertebrate cells including etoposide-treated fibroblasts, and gamma-irradiated thymocytes. Expression of a conditional p53-fusion protein demonstrates that activation of wild-type p53 function in E1A and T24 H-ras-transformed p53-deficient fibroblasts is sufficient for induction of the EI24 mRNA.
The novel EI24 gene and the corresponding EI24 protein of mammals have thus been isolated and characterized, and are described in a number of embodiments herein. The present invention thus relates to an apoptosis associated protein EI24, products and processes involved in the cloning, preparation and expression of genes for EI24; antibodies with specificity to EI24; and nucleotide probes corresponding to the EI24 nucleotide sequence or portions thereof. The EI24 polypeptide is useful for producing antibodies thereto. The antibodies and probes are useful for detecting and isolating EI24 in biological specimens including for example, cells from all human tissues including heart tissue, lung tissue, tumor cells, brain tissue, placenta, liver, skeletal muscle, kidney, and pancreas.
The present invention further relates to species homologs and viral homologs of EI24.
In a particular embodiment, the human EI24 gene has been cloned. The present invention thus relates to the cloning, identification, characterization and sequencing of cDNAs and genomic fragments which encode the EI24 that is present in human cells. The present invention further relates to a method for isolating EI24 partial clones using polymerase chain reaction (PCR) cloning, from diverse human tumor cell lines.
According to the present invention, there are provided genetic sequences encoding EI24. The instant invention also provides for expression vectors containing such genetic sequences, hosts transformed with such expression vectors, and methods for producing the genetically engineered or recombinant EI24.
The present invention also provides antibodies which specifically recognize EI24.
The EI24 cDNA and recombinant protein are useful for making antibodies which specifically recognize EI24. Such antibodies are useful for detecting and isolating EI24 in a biological specimen. The EI24 protein is also useful as a mediator of p53 function, particularly, p53 tumor suppression function, including p53 mediated apoptotic and/or cell cycle control function.
The present invention is further directed to methods for inducing or suppressing p53 mediated functions including apoptosis in individuals suffering from degenerative disorders Characterized by inappropriate cell proliferation or inappropriate cell death, respectively. Degenerative disorders characterized by inappropriate cell proliferation include, for example, inflammatory conditions, cancer, including lymphomas, genotypic tumors, etc. Degenerative disorders characterized by inappropriate cell death include, for example, autoimmune diseases, acquired immunodeficiency disease (AIDS), cell death due to radiation therapy or chemotherapy, etc.
The present invention also relates to methods for detecting the presence of EI24 protein, as well as methods directed to the diagnosis of degenerative disorders, which disorders are associated with an increased or decreased level of expression of EI24, as compared to the expected level of EI24 expression in the normal cell population.
The present invention is further directed to methods for monitoring the progress of degenerative disorders associated with increased or decreased levels of expression of EI24, by monitoring EI24 expression.
The present invention also relates to methods for determining whether a disease/degenerative disorder is linked to abnormal EI24 expression, as well as methods for determining the effect of over expression or loss of expression of EI24 in animal models such as transgenic mice and/or homozygous null mice. Methods for determining whether a disease/degenerative disorder is linked to abnormal EI24 expression include analyzing EI24 expression in diseased tissue as compared to normal tissue by for example, Northern and/or Western blots, as well as by other assay methods readily chosen and employed by those of ordinary skill in the art.
The present invention also relates to therapeutic methods and compositions for modulating apoptotic effects by administering EI24 protein, or a mutant or hybrid thereof, or by modulating expression of the EI24 gene, to an individual suffering from a degenerative disorder characterized by inappropriate cell proliferation or inappropriate-cell death in order to stabilize inappropriate cell proliferation (i.e., induce apoptosis) or stabilize inappropriate cell death (i.e., suppress apoptosis), respectively, and/or in either case to restore normal cell behavior.
The present invention further relates to functional equivalents including functional fragments of EI24 and/or EI24.
The present invention is also directed to nucleotide probes which can be used to determine the presence of EI24 as well as to identify and isolate homologs including species homologs and viral homologs.